Mouse TNF alpha ELISA Kit
- Product Type:
- ELISA Kit
- 96 Assays
- ELISA Type:
- Sandwich ELISA, Double Antibody
- TNFalpha, Tumor Necrosis Factor Alpha, TNF-alpha, DIF, TNF-alpha, TNFA, TNFSF2
|Product Name:||Mouse TNF alpha ELISA Kit|
|Alias:||TNFalpha, Tumor Necrosis Factor Alpha, TNF-alpha, DIF, TNF-alpha, TNFA, TNFSF2|
|Detection Method:||Sandwich ELISA|
|Application:||This immunoassay kit allows for the in vitro quantitative determination of Mouse TNF-alpha concentrations in serum plasma and other biological fluids.|
|Storage:||4°C for 6 months|
|Note:||For Research Use Only|
|Recovery:||Matrices listed below were spiked with certain level of Mouse TNF-alpha and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse TNF-alpha in samples.|
|Linearity:||The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse TNF-alpha and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.|
|Intra Assay:||CV <8%|
|Inter Assay:||CV <10%|
|ELISA Microplate (Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody(Concentrated)||120ul||4°C (Protect from light)|
|Antibody Dilution Buffer||10ml||4°C|
|HRP-Streptavidin Conjugate(SABC)||120ul||4°C (Protect from light)|
|SABC Dilution Buffer||10ml||4°C|
|TMB Substrate||10ml||4°C (Protect from light)|
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
|UniProt Protein Function:||TNF-a: Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Homotrimer. Interacts with SPPL2B. Belongs to the tumor necrosis factor family.|
|UniProt Protein Details:|
Protein type:Motility/polarity/chemotaxis; Membrane protein, integral; Apoptosis; Cytokine
Cellular Component: extracellular space; cell surface; integral to plasma membrane; extracellular region; integral to membrane; lipid raft; secretory granule; recycling endosome; membrane; plasma membrane; intracellular; phagocytic cup; external side of plasma membrane
Molecular Function:identical protein binding; protein binding; protease binding; cytokine activity; tumor necrosis factor receptor binding
Biological Process: positive regulation of JNK activity; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; positive regulation of NFAT protein import into nucleus; activation of MAPK activity; positive regulation of osteoclast differentiation; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; multicellular organismal development; response to glucocorticoid stimulus; positive regulation of caspase activity; positive regulation of NF-kappaB import into nucleus; osteoclast differentiation; positive regulation of translational initiation by iron; positive regulation of membrane protein ectodomain proteolysis; activation of NF-kappaB transcription factor; positive regulation of MAP kinase activity; tumor necrosis factor-mediated signaling pathway; cellular extravasation; positive regulation of phagocytosis; negative regulation of interleukin-6 production; JNK cascade; negative regulation of osteoblast differentiation; positive regulation of action potential; regulation of immunoglobulin secretion; negative regulation of protein complex disassembly; positive regulation of cytokine production; positive regulation of heterotypic cell-cell adhesion; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of mitosis; response to virus; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; glucose metabolic process; positive regulation of chemokine production; negative regulation of cytokine secretion during immune response; positive regulation of protein transport; detection of mechanical stimulus involved in sensory perception of pain; cell activation; defense response to Gram-positive bacterium; organ morphogenesis; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; defense response to bacterium; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; negative regulation of L-glutamate transport; negative regulation of transcription, DNA-dependent; leukocyte migration; sequestering of triacylglycerol; apoptosis; positive regulation of smooth muscle cell proliferation; positive regulation of JNK cascade; defense response; negative regulation of transcription from RNA polymerase II promoter; positive regulation of interleukin-18 production; signal transduction; chronic inflammatory response to antigenic stimulus; positive regulation of synaptic transmission; positive regulation of hair follicle development; negative regulation of cell proliferation; regulation of protein secretion; regulation of osteoclast differentiation; negative regulation of lipid catabolic process; positive regulation of neuron apoptosis; positive regulation of cell proliferation; lipopolysaccharide-mediated signaling pathway; protein kinase B signaling cascade; positive regulation of chronic inflammatory response to antigenic stimulus; regulation of I-kappaB kinase/NF-kappaB cascade; inflammatory response; caspase activation; positive regulation of humoral immune response mediated by circulating immunoglobulin; regulation of protein amino acid phosphorylation; positive regulation of protein complex disassembly; transformed cell apoptosis; calcium-mediated signaling; MAPKKK cascade; positive regulation of peptidyl-serine phosphorylation; humoral immune response; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; cell proliferation; positive regulation of interferon-gamma production; negative regulation of glucose import; positive regulation of programmed cell death; positive regulation of chemokine biosynthetic process; positive regulation of protein complex assembly; negative regulation of viral genome replication; protein import into nucleus, translocation; positive regulation of protein kinase activity; activation of MAPKKK activity; positive regulation of fever; immune response; positive regulation of protein amino acid phosphorylation; receptor biosynthetic process; negative regulation of myoblast differentiation; leukocyte tethering or rolling; regulation of insulin secretion; positive regulation of cytokine secretion; positive regulation of inflammatory response
|NCBI Summary:||This gene encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. Members of this family are classified based on primary sequence, function, and structure. This protein is synthesized as a type-II transmembrane protein and is reported to be cleaved into products that exert distinct biological functions. It plays an important role in the innate immune response as well as regulating homeostasis but is also implicated in diseases of chronic inflammation. In mouse deficiency of this gene is associated with defects in response to bacterial infection, with defects in forming organized follicular dendritic cell networks and germinal centers, and with a lack of primary B cell follicles. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jun 2013]|
|NCBI GenInfo Identifier:||135935|
|NCBI Gene ID:||21926|
|UniProt Secondary Accession:||P06804,O35853, Q62326, Q91VF3,|
|UniProt Related Accession:||P06804|
|Molecular Weight:||25,896 Da|
|NCBI Full Name:||Tumor necrosis factor|
|NCBI Synonym Full Names:||tumor necrosis factor|
|NCBI Official Symbol:||Tnf|
|NCBI Official Synonym Symbols:||DIF; Tnfa; TNF-a; TNFSF2; Tnfsf1a; TNFalpha; TNF-alpha|
|NCBI Protein Information:||tumor necrosis factor; cachectin; tumor necrosis factor-alpha; tumor necrosis factor ligand superfamily member 2|
|UniProt Protein Name:||Tumor necrosis factor|
|UniProt Synonym Protein Names:||Cachectin; TNF-alpha; Tumor necrosis factor ligand superfamily member 2; TNF-a|
|Protein Family:||Tumor necrosis factor|
|UniProt Gene Name:||Tnf|
|UniProt Entry Name:||TNFA_MOUSE|
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
|1.||Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!|
|2.||Aliquot 0.1ml standard solutions into the standard wells.|
|3.||Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.|
|4.||Add 0.1 ml of properly diluted sample (Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.|
|5.||Seal the plate with a cover and incubate at 37 °C for 90 min.|
|6.||Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.|
|7.||Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.|
|8.||Seal the plate with a cover and incubate at 37°C for 60 min.|
|9.||Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.|
|10.||Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.|
|11.||Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.|
|12.||Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.|
|13.||Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.|
|14.||Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.|
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
|Urine & Cerebrospinal Fluid:|| |
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
|Cell culture supernatant:|| |
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
|Cell lysates:|| |
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
|Tissue homogenates:|| |
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
|Tissue lysates:|| |
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
|Breast Milk:|| |
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.